U.S. patent number 5,756,323 [Application Number 08/244,378] was granted by the patent office on 1998-05-26 for method for generating structural and functional diversity in a peptide sequence.
This patent grant is currently assigned to Institut Pasteur. Invention is credited to Noelle Doyen, Sacha Kallenbach, Francois Rougeon.
United States Patent |
5,756,323 |
Kallenbach , et al. |
May 26, 1998 |
Method for generating structural and functional diversity in a
peptide sequence
Abstract
The invention relates to a method for generating structural and
functional diversity in a peptide sequence by randomly deleting and
inserting nucleotides in a nucleotide sequence which codes for the
peptide sequence. This method may be carried out by transfecting a
cell preparation with vectors allowing expression of the Rag-1 and
Rag-2 genes and optionally the terminal deoxynucleotidyl
transferase gene, as well as with a vector including the nucleotide
sequence which codes for the peptide sequence.
Inventors: |
Kallenbach; Sacha (Nanterre,
FR), Doyen; Noelle (Paris, FR), Rougeon;
Francois (Poigny La Foret, FR) |
Assignee: |
Institut Pasteur (Paris Cedex,
FR)
|
Family
ID: |
9419935 |
Appl.
No.: |
08/244,378 |
Filed: |
September 1, 1994 |
PCT
Filed: |
December 11, 1992 |
PCT No.: |
PCT/FR92/01178 |
371
Date: |
September 01, 1994 |
102(e)
Date: |
September 01, 1994 |
PCT
Pub. No.: |
WO93/12228 |
PCT
Pub. Date: |
June 24, 1993 |
Foreign Application Priority Data
|
|
|
|
|
Dec 11, 1991 [FR] |
|
|
91/15389 |
|
Current U.S.
Class: |
435/6.16;
435/465; 435/69.6; 536/23.2 |
Current CPC
Class: |
C07K
14/4705 (20130101); C07K 16/00 (20130101); C12N
9/00 (20130101); C12N 9/1264 (20130101); C12N
15/1024 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/47 (20060101); C07K
16/00 (20060101); C12N 9/00 (20060101); C12N
15/10 (20060101); C12N 9/12 (20060101); A61K
38/00 (20060101); C12N 015/01 (); C12N
015/12 () |
Field of
Search: |
;435/172.3
;536/23.2 |
Other References
Watson et al. Molecular Biology of the Gene, Fourth Editior pp.
313-338 Benjamin Cummings Pub. Co. (1987). .
Kallenbach et al. Proc. Natl. Acad. Sci. USA 89 2799-2803 (1992)
Three Lymphoid Specific Factors Account for all . . . .
Oettinger et al. Science 248 1517-1523 (1990). .
Rag-1 and Rag-2, Adjacent Genes that Synergistically . . . .
Kallenbach et al. Nucleic Acids Research 18 6730 (1990) A Rapid
Test for U(O)J Recombinase Activity. .
Kowai et al. Nucleic Acids Research 14 5777-5792 (1986) Isolation
and Characterization of Bovine and Mouse Terminal . . . .
Landau et al. Molecular and Cellular Biology 7 3237-3243 (1987)
Increased Frequency of N-region Insertion in a Murine Pre-B-cell .
. . . .
Huse et al. Science 246 1275-1281 (1989) Generation of a Large
Combinatorial Library of the Immunoglobulin . . . . .
Davis Annual Review of Biochemistry 59 475-496 (1990) T Cell
Receptor Gene Diversity and Selection. .
Hasty et al. Human neutrophil collagenase J. Biol. Chem. vol. 265
11421-11424, 1990..
|
Primary Examiner: Ketter; James
Assistant Examiner: Brusca; John S.
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier
& Neustadt, P.C.
Claims
We claim:
1. An isolated nucleic acid comprising nucleotide sequences
including the Rag- 1 and Rag-2 genes and the gene which codes for
terminal deoxymucleotidyl transferase (TdT).
2. The isolated nucleic acid according to claim 1, wherein the
nucleotide sequences are included in vectors.
3. A composition comprising the plasmids pBluerec, pRag-1, pRag-2,
and pMTdT.
4. A composition comprising a combination of synergistic quantities
of the products of expression of the Rag-1 and Rag-2 genes and a
terminal deoxynucleotide transferase.
5. A method for generating structural diversity in a peptide
sequence by randomly deleting or inserting nucleotides in a
nucleotide sequence which codes for the peptide sequence, said
method comprising the transfection of a mammalian cell preparation
with one or more vectors expressing the products of the Rag-1 gene,
Rag-2 gene and the terminal deoxynucleotidyl transferase (TdT) gene
and by an identical or different vector including said nucleotide
sequence bordered by one or more recombination signal sequences
(RSS) to produce a recombined vector or vectors that express said
nucleotide sequence to produce a mutated peptide.
6. A method for generating structural diversity in a peptide
sequence by randomly deleting or inserting nucleotides in a
nucleotide sequence which codes for the peptide sequence, the
method comprising the transfection of a mammalian cell preparation
with a vector including the nucleotide sequence bordered by one or
more RSS recombination sequences, then in a second step
transfection by one or more identical or different vectors
expressing the products of the Rag-1 gene, Rag-2 gene and terminal
deoxynucleotidyl transferase gene to produce a recombined vector or
vectors that express said nucleotide sequence to produce a mutated
peptide.
7. A method for generating structural diversity in a peptide
sequence by introducing into a nucleotide sequence corresponding to
the peptide sequence insertions or deletions resulting from the
inverse repetition of sequences adjacent to one or more RSS
recombination sequences, said method comprising the transfection of
a mammalian cell preparation with one or more vectors expressing
the products of the Rag-1 and Rag-2 genes and by an identical or
different vector including the nucleotide sequence bordered by one
or more RSS sequences to produce a recombined vector or vectors
that express said nucleic sequence to produce a mutated
peptide.
8. A method for generating structural diversity in a peptide
sequence by introducing into a nucleotide sequence corresponding to
the peptide sequence insertions or deletions resulting from the
inverse repetition of sequences adjacent to one or more RSS
recombination sequences, said method comprising the transfection of
a mammalian cell preparation with a vector including the nucleotide
sequence bordered by one or more RSS sequences or by one, then in a
second step transfection by one or more identical or different
vectors expressing the products of the Rag-1 and Rag-2 genes to
produce a recombined vector or vectors that express said nucleotide
sequence to produce a mutated peptide.
9. The method according to one of claims 5 to 8, wherein the
recombined vector or vectors including a nucleotide sequence
corresponding to the peptide sequence are transferred into bacteria
in order to select proteins showing a required structure.
10. The method according to one of claims 5 to 8, for obtaining
structurally diverse immunoglobulins by separate rearrangement of
genes encoding light and heavy chains and co-expression of the two
chains.
11. The method according to one of claims 5 to 8, for obtaining
structurally diverse receptors of lymphoid cells by rearrangement
of genes encoding the alpha, beta, gamma and/or delta chains of the
T cell receptors.
12. The method according to claim 10, wherein the rearrangement of
genes encoding the light chains is carried out in the presence of
the nucleotide sequences of the Rag-1 and Rag-2 genes.
13. The method according to claim 10, wherein the rearrangement of
genes encoding the heavy chains is carried out in the presence of
nucleotide sequences of the Rag-1 and Rag-2 genes and the gene for
the terminal deoxynucleotidyl transferase.
14. The method according to one of claims 5 to 8, comprising the
steps of:
a) transfection of a mammalian cell preparation with one or more
vectors including a nucleotide sequence which codes for the peptide
sequence for which the variability is desired and by one or more
identical or different vectors expressing the Rag-1 and Rag-2 genes
or expressing the Rag-1, Rag-2 and the TdT genes;
b) isolation of the DNA of the vectors of the cell
preparations;
c) removal of the vectors which have not undergone
recombination;
d) transformation of the cell hosts by the vectors resulting from
step c), and
e) selection of the cells expressing the molecules showing a
desired structure.
15. The method according to claim 14, wherein the said peptide
sequence is an alpha, beta, gamma or delta chain of the lymphoid
cell receptors.
16. The method according to claim 10, comprising the steps of:
a) co-transfection of a mammalian cell preparation with one or more
vectors including non-rearranged nucleotide sequences which code
for light chains and by one or more vectors expressing the Rag-1
and Rag-2 genes, and
b) co-transfection of another cell preparation by one or more
vectors including the non-rearranged nucleotide sequences which
code for heavy chains and by one or more vectors expressing the
gene for the terminal deoxynucleotidyl transferase as well as the
Rag-1 and Rag-2 genes,
c) isolation of the vector DNA of the two cell preparations,
d) removal of the vectors which have not undergone
recombination,
e) transformation of at least two bacterial cultures, each by one
of the two isolated vector DNAs obtained from the two cell
preparations obtained from step d),
f) amplification and preparation of the vector DNA,
g) insertion of the genes which code for the heavy and light chains
into the same vector,
h) transformation of cells by the vector obtained from step f),
and
i) selection of cells expressing the complete immunoglobulin
molecules.
17. The method according to claim 16, wherein the vectors obtained
at step c) that have not undergone recombination are removed by
enzymatic digestion.
18. The method according to claim 16, wherein the preparations are
fibroblasts or any other mammalian cells.
19. Plasmid pMTdT including the gene for the terminal
deoxyribonycleotidyl transferase deposited with the Microorganism
Culture National Collection under the N. I 1160.
20. The method according to claim 16, wherein the vector expressing
the terminal deoxyribonucleotidyl transferase is the plasmid pMTdT
deposited with the Microorganism Culture National Collection under
the No. I 1160.
Description
The present invention relates to a method for generating structural
or functional diversity in a peptide sequence by introducing
insertions or deletions of nucleotides in the nucleotide sequence
which codes for the said peptide sequence.
The present invention also relates to pharmaceutical compositions,
drugs or diagnostic reagents containing proteins or peptides
obtained by this method.
The mature genes coding for the constituent chains of
immunoglobulins and T cell receptors are assembled early during
lymphocyte development from gene segments termed variability (V),
linking, or junction (J), and in some cases diversity (D).
Seven loci are able to be rearranged by recombination of these
fragments.
The recombination signal sequences (RSS) adjacent to each gene
supply the targets for the recombination. These sequences are
composed of a palindromic heptamer and a nonamer rich in adenosine
and thymidine, separated by a sequence of 12 or 23 base pairs. The
rearrangements are made between RSS with separation sequences of
different lengths.
Two types of junction or linking are formed during the
recombination: coding junctions created by the juxtaposition of
gene segments and noncoding junctions formed by contiguous RSS. In
the latter case, the heptamers are generally joined without
nucleotide insertions or deletions. The coding junctions themselves
are liable to substantial modifications.
The variations in the junctions during the rearrangement of the
gene segments coding for the immunoglobulins represent a major
source of diversity. Several nucleotides can thus be eliminated and
two types of insertion can occur.
The random addition of nucleotides results in the insertion of
regions termed N regions in the immunoglobulin heavy chains. The
hypothesis has been advanced (Randau et al., Molecular and Cellular
Biology, 1987, 3237-3243) that the deoxynucleotidyl transferase
(TdT) is responsible for this random addition of nucleotides.
The type P nucleotides insertions correspond to the inverse
repetition of sequences adjacent to those of the coding sequences.
The hypothesis has been advanced that their addition represents a
necessary step in the recombination mechanism.
Transfection experiments with genomic DNA have enabled the
isolation of two genes actively involved in the recombination: the
Rag-1 and Rag-2 genes (Oettinger et al., Science, Volume 248,
1517-1523, 1990).
It has been shown that the Rag-1 and Rag-2 genes are responsible
for the site-specific recombination.
Nevertheless, the combination of the products of the Rag-1 and
Rag-2 genes does not restore the diversity of antibodies found in
vivo, in other words does not allow to add the N sequences.
Various methods have been developed to attempt to modify the
immunoglobulin heavy and light chains or the receptors.
EP patent No-368.684 relates to a method for cloning nucleotide
sequences corresponding to the variable regions of the molecules in
the immunoglobulin family. This method consists of producing a DNA
complementary to the variable region of the immunoglobulin.
EP patent No-328.444 relates to a method for modifying the
structure of an antibody while retaining its functional
specificity. Thus, the constant regions in particular are modified
by a classical genetic engineering technique.
To the knowledge of the applicant, there is no method for
efficiently obtaining structurally modified immunoglobulins with a
wide diversity in the modifications.
The applicant has thus aimed to demonstrate recombination
mechanisms which allow the organism to achieve a wide diversity in
the immunoglobulins such as the IgG, the IgM, the IgA, the IgE, and
in the lymphoid cell receptors.
The applicant has also aimed to develop a general method for
randomly obtaining a very diversified range of mutations, in
particular by random insertion, in the nucleotide sequence
corresponding to proteins with various structures and
functions.
The applicant has also found that the combination of the products
of expression of the Rag-1 and Rag-2 genes leads unexpectedly to a
site-specific recombination and that the introduction of the TdT
leads to a junction diversity between the DJ and VDJ sequences
equivalent to those found in vivo.
The applicant has also shown that it is possible, by use of the
Rag-1 and Rag-2 products as well as the terminal deoxynucleotidyl
transferase, to obtain in vitro the production of antibodies
showing rearrangements, and which statistically show an extensive
structural and functional diversity.
The present invention thus relates to a composition containing a
combination of synergistic quantities of the products of expression
of the Rag-1 and Rag-2 genes and a terminal deoxynucleotidyl
transferase or one or more of their biologically active
fragments.
It also relates to a composition containing the nucleotide
sequences including the Rag-1 and Rag-2 genes or the genes leading
to the synthesis of fragments or biologically active derivatives of
the products of the Rag-1 and Rag-2 genes and the gene coding for
the TdT or one of its fragments or biologically active derivatives,
in which the nucleotide sequences are advantageously carried by
vectors.
It in addition relates to a composition comprising the plasmids p
Blue Rec (Kallenbach et al., Nucleic Acid Research, 18, 6730,
1990), p Rag-1 and p Rag-2 (Oettinger et al, previously referred
to).
The present invention also relates to
a method for generating structural or functional diversity in a
peptide sequence by randomly deleting or inserting nucleotides in a
nucleotide sequence which codes for this peptide sequence, the said
method comprising the transfection of a cell preparation with one
or more vectors allowing expression of the products of the Rag-1,
Rag-2 genes and the terminal deoxynucleotidyl transferase (TdT) or
of their derivatives and by an identical or different vector
including the said nucleotide sequence bordered by one or more RSS
recombination sequences or bordered by one or more biologically
active derivatives of the RSS sequences.
a method for generating structural or functional diversity in a
peptide sequence by randomly deleting or inserting nucleotides in a
nucleotide sequence which codes for this peptide sequence, the said
method comprising the transfection of a cell preparation with a
vector including the said nucleotide sequence bordered by one or
more RSS recombination sequences or bordered by one or more
biologically active derivatives of the RSS sequences, then in a
second step by one or more identical or different vectors allowing
expression of the products of the Rag-1, Rag-2 genes and terminal
deoxynucleotidyl transferase or of their derivatives.
a method for generating structural or functional diversity in a
peptide sequence by introducing into the nucleotide sequence
corresponding to this peptide sequence insertions or deletions
resulting from the inverse repetition of sequences adjacent to the
RSS recombination sequences, the said method comprising the
transfection of a cell preparation with one or more vectors
allowing expression of the products of the Rag-1 and Rag-2 genes or
of their derivatives and by an identical or different vector
including the said nucleotide sequence bordered by one or more RSS
sequences or by one or more biologically active derivatives of the
RSS sequences.
a method for generating structural or functional diversity in a
peptide sequence by introducing into the nucleotide sequence
corresponding to this peptide sequence insertions or deletions
resulting from the inverse repetition of sequences adjacent to the
RSS recombination sequences, the said method comprising the
transfection of a cell preparation with a vector including the said
nucleotide sequence bordered by one or more RSS sequences or by one
or more biologically active derivatives of the RSS sequences, then
in a second step by one or more identical or different vectors
allowing expression of the products of the Rag-1 and Rag-2 genes or
of their derivatives.
Such methods also allow the random introduction of insertions and
deletions into nucleotide sequences. These sequence modifications
thus allow the biological diversity to be increased at will.
Such methods are in particular interesting substitutes for the
traditional methods of mutagenesis and for methods of obtaining
monoclonal antibodies by hybridomas.
The possibility of obtaining monoclonal antibodies by a method
other than from hybridomas is advantageous in human therapeutics
since the antibody preparations obtained according to the invention
are pure.
It should be remembered that N sequences mean random insertions of
nucleotides, or in other words those which are not replicas of
sequences already existing in the neighborhood of the RSS.
These N sequences are thus created randomly and as a result show a
very wide diversity, which is not dependent on the nucleotide
sequence in the neighborhood of the RSS.
The P sequences are on the other hand insertions resulting from the
inverse repetition of sequences adjacent to the RSS.
As a result, they show less diversity than the N regions.
Advantageously, the recombined vector or vectors including the
nucleotide sequence corresponding to the peptide sequence are
transferred into bacteria in order to select the proteins or
peptides showing the structure and/or function desired.
It should be noted that it is necessary to choose expression
vectors for the proteins or peptides which are suited to the cells,
eukaryotes or prokaryotes, used. It is also possible to use the
plasmids pcDNAI (marketed by In vitrogen) or pRc/CMV (marketed by
In Vitrogen) for the eukaryotic cells or the plasmid pBlue Script
(marketed by Stratagen)
The present invention also relates to a preferential use of the
method for obtaining immunoglobulins, in particular antibodies
showing a wide structural and functional diversity by separate
rearrangements of the light and heavy chains making up the
immunoglobulins and co-expression of the two chains in the same
cell.
Thus, this preferential mode of application allows the production
of a large quantity of cells expressing varied sequences of the two
immunoglobulin chains. A subsequent step allows the selection of
the specific immunoglobulin for a given pathogenic agent, for
example.
Advantageously, the sequence corresponding to the heavy chains used
only includes the Fab. part of these chains. The Fc part is added
later.
Preferentially, the rearrangement of the light chains is carried
out in the presence of the nucleotide sequences of the Rag-1 and
Rag-2 genes or of genes leading to the synthesis of biologically
active derivatives or fragments of the products of Rag-1 and Rag-2,
and the rearrangement of the heavy chains is carried out in the
presence of the nucleotide sequences of the Rag-1 and Rag-2 genes
and of the terminal deoxynucleotidyl transferase gene or of genes
leading to the synthesis of biologically active derivatives or
fragments of Rag-1, Rag-2 or the TdT.
In order to obtain the expression of the immunoglobulin heavy
chains, vectors including the V, D and J segments are used, while
for the light chains the vectors include the v and J sequences.
The present invention in addition relates to a method for obtaining
receptors of lymphoid cells, and in particular T cells, showing a
wide structural and functional diversity by rearrangement of the
alpha, beta, gamma and/or delta chains of the T cell receptors.
It also relates to a method comprising the steps of:
a) transfection of a cell preparation with one or more vectors
including a nucleotide sequence which codes for the peptide
sequence for which the variability is desired and by one or more
identical or different vectors allowing expression of the Rag-1 and
Rag-2 genes or expression of the Rag-1, Rag-2 genes and the
TdT;
b) isolation of the DNA of the vectors of the cell
preparations;
c) removal of the vectors which have not undergone
recombination;
d) transformation of the cell hosts by the vectors resulting from
step c), and
e) selection of the cell hosts expressing the molecules showing the
structure and/or function sought for.
Advantageously, this method comprises the steps of :
a) co-transfection of a cell preparation with one or more vectors
including the genes which code for the non-rearranged light chains
and by one or more vectors expressing the genes which code for
Rag-1 and Rag-2, their derivatives and/or their fragments, and
b) co-transfection of another cell preparation by one or more
vectors including the genes which code for the non-rearranged heavy
chains and by one or more vectors expressing the gene for the
terminal deoxynucleotidyl transferase in addition to the genes
coding for Rag-1 and Rag-2,
c) isolation of the vector DNA of the two cell preparations,
d) removal of the vectors which have not undergone
recombination,
e) transformation of at least two bacterial cultures respectively
by the vector preparations obtained from step
d), amplification and preparation of the bacterial vector DNA,
f) insertion of the genes which code for the heavy and light chains
into the same vector,
g) transformation of the cell hosts by the vector obtained from
step f), and
h) selection of the cell hosts expressing the complete
immunoglobulin molecules.
Cell hosts are here taken to mean any eukaryotic bacteria or cells
able to be transformed or transfected.
In the present application, vector is taken to mean any
autoreplicative DNA molecule able to be transferred from one cell
to another. Plasmids are preferentially used in steps a) to h) but
any other vector compatible with the cell and bacterial systems
used may advantageously be used.
The vectors obtained at step c) that have not undergone
recombination are advantageously removed by enzymatic digestion at
a site specifically recognized by a restriction endonuclease.
The cells preferentially used in steps a) and b) are fibroblasts or
lymphoid cells, or any other eukaryotic cell type or cell line.
It should be noted that the vectors used preferentially contain the
precocious region of the polyome in order to allow their
replication at the autonomous state of the eukaryotic cells.
The selection of the bacteria at step h) is advantageously carried
out by filter replication of the bacterial colonies obtained by
spreading the bacteria on Petri dishes and subsequent screening
["Molecular cloning ; a Laboratory Manual" (Sambrok et al., Cold
Spring Harbor Laboratory Press, New York, 1989)] with the antigens
for the required specific antibodies.
It should also be noted that the vectors expressing the antibodies
or the lymphoid cell receptors sought for may subsequently be
modified so as to allow the expression of complete immunoglobulins
in the eukaryotic cells, and in particular so as
The methodir glycosylation.
The methods by which steps a) to h) may be carried out are well
known to those skilled in the art. In general, "Molecular cloning;
a Laboratory Manual", (Sambrok et al., Cold Spring Harbor
Laboratory Press, New York, 1989) may be referred to.
Some practical applications of these steps are also described in
the article by Huse et al. (Science, volume 246, 1275-1281,
1989).
In particular, the vector used in step a) may be the plasmid p Blue
Script containing a cassette of the type fragment EcoRI-Not I of
lambda Lcl described in this article in which are inserted a V
segment and a J segment attached to the constant region of the
light chain. The VL and JL segments are bordered by their
recombination signal sequences.
The vector used for the co-transformation of step b) may be the
plasmid p Blue Script containing a cassette of the type fragment
NotI-EcoRI of lambda Hc2 described in this article in which are
inserted a V segment, a D segment and a J segment attached to the
CH1 region of the heavy chain. The VH, DH and JH segments are
bordered by their recombination signal sequences.
The expression vectors of the Rag-1 and Rag-2 genes used in steps
a) and b) may be those described by Qettinger et al. (Science, 248,
1517-1523, 1990).
The cloning vector including the gene which codes for the terminal
deoxyribonucleotidyl transferase may in particular be the plasmid
pMTdT which is a pcDNAII in which the complementary DNA of the
mouse terminal deoxyribonucleotidyl transferase has been
inserted.
The present application also relates to this plasmid, which was
deposited on 10 Dec. 1991 with the Microorganism Culture National
Collection of the Institut Pasteur under the n. I 1160.
The previously mentioned article by Huse et al. moreover mentions
some practical applications which may be used within the scope of
the present invention.
In particular, the vectors used in steps a) and b) may be obtained
from a library created as described in this article.
This library may be obtained by cloning the light and heavy chain
fragments in respectively the vectors generated from phage lambda,
lambda Lc1 and lambda Hc2. These vectors, which carry out the
cloning in the initial stage of the library creation, can be
excised to give rise to a plasmid containing oligonucleotide
fragments corresponding to the heavy and light chains.
These vectors contain various restriction sites, and a sequence
which codes for the leader peptide of the bacterial gene PelB which
has previously been successfully used in E. coli to secrete Fab
fragments, a ribosome binding site, and on the lambda Hc2 vector a
sequence corresponding to the tag decapeptide located at the C
terminal end of the insertion into the heavy chain. The tag peptide
enables the expression products to be purified by passage through
immunoaffinity columns.
The DNA used as the basis for creating the heavy chain library is
preferentially human DNA in order to minimize the risk of rejection
by the organism in the case of the use of these antibodies in human
therapeutics.
The use of step f), which is the insertion of the genes coding for
the heavy and light chains into the same vector, can be carried out
as described on page 1278 of the article by Huse et al. previously
referred to.
The library of light chains is thus digested by the restriction
endonuclease cleaving at a unique site, the resulting 5'
extremities are dephosphorylated, and the products are then
digested by another restriction endonuclease Eco R1 cleaving at a
unique site.
The DNA of the vectors constituting the library of heavy chains is
cleaved by the endonuclease Hind-III, then dephosphorylated and
digested by endonuclease Eco R1.
The DNA thus prepared are mixed and linked by ligation.
After ligation, only the clones which result from the combination
of a fragment derived from the library of heavy chains and a
fragment derived from the library of light chains result in a
viable phage.
For the creation of the libraries of heavy and light chains,
preparations of messenger DNA isolated from cells from the organism
or hybridomas may be used. The corresponding complementary DNA are
then synthesized in a PCR amplification system. These techniques
are well known to a person skilled in the art and are in particular
described in "Molecular cloning; a Laboratory Manual" (Sambrok et
al., 1989, previously referred to).
The selection of the bacteria expressing the complete molecules in
step h) is followed by a step for selecting the clones synthesizing
the required molecules.
The assembly of the parts of the heavy and light chains thus
obtained can lead uniquely to the production of the Fab fragment.
The vector is then modified so that it can code for the Fc
fragment. The expression product of this vector is thus an
antibody.
In order to select the antibody-synthesizing clones, the selection
method described by Huse et al. (previously referred to) for the
selection of clones synthesizing antibodies directed against
paranitrophenyl phosphonamidate (NPN) may be used.
The method used in this article consists of making duplicates on
nitrocellulose sheet of clones spread on dishes of gel culture
medium and testing the hybridization of NPN coupled with .sup.125
I-labeled bovine serum albumin.
The present invention also relates to pharmaceutical compositions,
drugs and diagnostic reagents containing products obtained by one
of the methods of the present invention.
In particular, the products from expression in eukaryotic or
prokaryotic cells transfected with recombinant plasmids including
genes of rabbit or mouse origin may be used in human or veterinary
diagnostic kits.
The present invention in addition relates to immunogenic
compositions and antibodies obtained by one of the methods of the
invention.
The present invention is illustrated without in any way being
restricted by the following examples of its application in
which:
FIG. 1 represents the sequences of junctions (SEQ ID NOS: 1-18)
formed on the pBlueRec plasmid after co-transfection with p Rag-1
and p Rag-2 in NIH-3T3 fibroblasts.
FIG. 2 represents the sequences of junctions (SEQ ID NOS: 1, 19-51)
formed on the pBlueRec plasmid after co-transfection with the
vectors coding for Rag-1, Rag-2 and the TdT in NIH-3T3
fibroblasts.
FIG. 3 represents the sequences of junctions (SEQ ID NOS: 1, 52-62)
formed on pBlueRec after co-transfection with the vectors coding
for Rag-1 and Rag-2 in the cell lines BW1J, CHO-K1 and A.9.
In these three figures, the sequences of the recombined plasmids
are aligned with the sequence of the original pBlueRec plasmid,
which is shown at the top of each figure.
In these three figures, the nucleotides presumed to be due to type
P insertions are underlined in the central parts of the figures,
while the type N insertions shown in the same parts are not
underlined. The deletions are represented by broken lines.
EXAMPLES
Materials and methods used in these examples
Cell line
The NIH-3T3 fibroblasts from mouse embryos (ATCC CRL 6442) and the
A9 cells derived from L cells (ATCC CRL 6319) were cultivated in
DMEM supplemented with 10% of calf foetus serum.
The BW1J mouse hepatoma cells were cultivated as described by
Cassio D. and Weiss M. C. (Somat Cell Genet, 5, 719-738, 1979). The
CHO-K1 Chinese hamster ovarian cells (ATCC CCL 61) were cultivated
in RPMI supplemented with 10% of calf foetus serum.
The BASP-1 pre-B cells (Choquet et al., Science, 235, 1211-1214,
1987) were cultivated in RPMI supplemented with 10% of calf foetus
serum and 50 pm of 9-mercapto-ethanol.
Cloninc of the aene coding for the mouse terminal deoxvnucleotidvl
transferase.
The RNA was prepared from the thymuses of five-week-old mice as
described by Auffray and Rougeon (Europe J. Biochem., 107, 303-314,
980), but using 4M guanidine thiocyanate in place of the 6M
urea.
The poly-A RNA was purified by use of oligo DT cellulose
chromatography and analyzed by Northern blot. The synthesis of the
single strand was carried out with 5 .mu.g of poly-A RNA initiated
with oligo DT using the MMLV inverse transcriptase (marketed by
BRL).
The double strand synthesis was carried out in the presence of DNA
polymerase I and RNase H.
The double-strand adapters with BstX1 terminals were linked to
suitably prepared CDNA and cloned in the BstX1 restriction site of
pCDNA2 (marketed by In Nitrogen).
The library of mouse thymus complementary DNA was screened with two
mixed oligonucleotides corresponding respectively to the sequences
121-142 and 1471-1494 of the mouse TdT complementary DNA
sequence.
The positive complementary DNA clones which were assumed to be
sufficiently long to contain the whole of the gene coding for the
mouse TdT were sequenced onto the two strands by the dideoxy
termination method (Sanger et al., PNAS, 74, 5463-5467, 1977).
Vectors used.
The pBlueRec plasmid was as described by Kallenbach et al. [(1990)
Nucleic Acid Research 18, 6730].
p Rag-1 and p Rag-2 were supplied by Qettinger et al. (previously
referred to).
The complementary DNA from the mouse TdT was cloned in the pCDNAl.
The expression of the Rag-1, Rag-2 and the TdT were under the
control of the CMV promoter.
Demonstration of the specific recombination
The transfections were carried out by electroporation following the
procedure described by Chu et al. (Nucleic Acid Research Res., 15,
1311-1326, 1987).
2.10.sup.6 cells were transfected with 2.5 .mu.g of pBlueRec with
or without 6 .mu.g of p Rag-1 or 4.8 .mu.g of p Rag-2.
In order to determine the effect of the TdT on the N region
insertion, 4.5 .mu.g of the TdT expression vector were added to the
three vectors previously mentioned.
The cells were collected after 40 to 48 hours incubation at
37.degree. C., washed with PBS and the plasmid DNA was prepared
according to Birnboim and Doly (Nucleic Acids Res. 7, 1513-1523,
1979).
The DNA sediments were resuspended in 20 .mu.l of sterile water. 7
.mu.l of the DNA solution were digested by Dpn1 in order to
eliminate the plasmids which had not replicated. 40 .mu.l of
XL1-Blue competent bacteria (marketed by Stratagene) were
transformed by electroporation and spread on LB Agar dishes
containing XGal (80 .mu.g/ml), IPTG (150 .mu.M), ampicillin (100
.mu.g/ml) and tetracycline (10 .mu.g/ml).
The rearrangement frequency was calculated as the quantity of blue
colonies.times.3 divided by the total number of clones.
Recombinant clone seauencina
The blue colonies were subcultured and isolated on LB Agar dishes
containing XGal, IPTG, ampicillin and tetracycline.
The DNA preparations were carried out according to the method
described by Sambrok et al. (Molecular cloning, Laboratory Manual
previously referred to), then treated with RNase for two hours at
room temperature before carrying out the double-strand
sequencing.
Example 1
Comparison of the recombination frequencies in the NIH-3T3
fibroblasts and in the B cell precursor cell lines in the presence
of Raa-1 and Raa-2.
The recombinant activity caused by Rag-1 and Rag-2 in the NIH-3T3
fibroblasts was tested by transient transfection.
p Rag-1 and p Rag-2 were co-transfected in the NIH3 fibroblasts
with the pBlueRec plasmid recombination substrate.
After 48 hours, the plasmid DNA was isolated and tested in E. coli
for recombination.
The LacZ sequence of pBlueRec was interrupted by a DNA fragment of
280 base pairs bordered by two RSS.
The site-specific rearrangement eliminated the insertion and in one
out of three cases restored the correct-reading framework, giving
rise to blue clones after transformation of E. Coli.
This rapid test enabled a substantial number of rearrangements to
be examined.
The transfection experiments with p Rag-1 or p Rag-2 alone did not
give rise to recombinant clones.
As shown in Table I, a significant recombination frequency was
observed when the two plasmids were co-transfected. In addition the
frequency (geometric mean=1.26) was comparable with that observed
after transfection of the recombination substrate in the pre B
cells, BASP1 (geometric mean=1.46).
In order to compare the coding junctions formed after the
recombination modulated in the fibroblasts by p Rag-1 and p Rag-2,
with the junctions observed in the lymphoid cells, the junctions in
the rearranged plasmids obtained after transfection of the NIH-3T3
cells were sequenced.
The sequences shown in FIG. 1 represent independent recombination
data, i.e. the results of different transfection experiments.
Seven out of 17 junctions showed neither insertions nor
deletions.
Four junctions had deletions on one side and four others had
deletions on both sides.
Only one junction showed a type P insertion of two base pairs
associated with a deletion of two base pairs on one side of the
junction.
The last junction showed a deletion of one base pair and an
addition of one nucleotide, which can be attributed to the heptamer
and is probably due to an imprecise excision.
Example 2
Co-transfection with Rac-1 and Raa-2 and TdT of the NIH-3T3
fibroblasts.
In order to attempt to reconstitute the junctional diversity
observed in the pre-B or pre-T cells, the NIH-3T3 fibroblasts were
transfected with the expression vector of the TdT as well as with p
R-1, p Rag-2 and pBlueRec.
The recombinant plasmids were sequenced.
Control transfections carried out with the pCDNA1 vector without
the complementary DNA from the TdT did not lead to any insertion of
N regions.
As shown in FIG. 2, 88% of the junctions revealed that there had
been N region insertions. The majority of N regions were from 1 to
4 nucleotides with an average of 3 nucleotides per junction.
Nevertheless an unusual insertion of 18 nucleotides was
observed.
The TdT incorporated the G residues more efficiently than the other
nucleotides. The frequency of type P nucleotide insertion seemed to
have increased in this experiment. It should however be noted that
it is impossible to distinguish them from the N regions.
Example 3
Effect of Rag-1 and Rag-2 in different types of differentiated cell
lines.
In the preceding examples it has been shown that Rag-1 and Rag-2
are capable of causing a recombination activity in the relatively
undifferentiated cells NIH-3T3 fibroblasts.
The activity of these two genes was thus tested in cells in
differentiated states. The results obtained in the BW1J, CHO-K1 and
A9 cell lines are shown in table 2.
Variations between the different cell lines can be seen, but
surprisingly the rearrangement frequencies in the BWLJ and CHO-K1
lines were clearly higher than those obtained for the 3T3
fibroblasts.
The rearrangements could be detected 13 hours after transfection
with pBlueRec, p Rag-1 and p Rag-2.
The sequencing of the recombined plasmids showed that deletions at
the coding junctions had taken place in the three cell lines tested
(FIG. 3).
A single type P nucleotide insertion was found at the junction of a
recombinant clone obtained after transfection of the A9 lines.
CONCLUSIONS
The results overall show that nucleotide deletions are obtained in
undifferentiated lines after co-transfection with p R-1 and p
Rag-2. In addition type P nucleotide insertions such as those
defined by Lafaille et al. (Cell, 59, 859-870, 1989) were
observed.
It was moreover observed that the co-expression of Rag-1, Rag-2 and
TdT in the undifferentiated cells leads to type N insertions.
These results thus show that Rag-1 and Rag-2 are sufficient to
induce a site-specific recombination but that the presence of the
TdT, in combination with R-1 and Rag-2, is necessary for obtaining
type N insertions which are a reflection of the expression of the
diversity of the synthesis of the immunoglobulins and the lymphoid
cell receptors.
TABLE I ______________________________________ Rearrangements in
the fibroblasts and the B cell precursors Amp.sup.R colonies R = 3
.times. Blue .times. 10.sup.-2 Cell line DNA Total Blue Total
______________________________________ NIH-3T3 pBlueRec 70,000 0 0
pBlueRec, pRag1 34,860 0 0 pBlueRec, pRag2 12,140 0 0 pBlueRec,
pRag1, 144,000 183 0.38 pRag2 pBlueRec, pRag1, 38,000 128 1 pRag2
pBlueRec, pRag1, 60,000 1059 5.3 pRag2 BASP-1 pBlueRec 14,400 186
1.3 pBlueRec 14,800 237 1.6 pBlueRec 12,960 190 1.5
______________________________________ R = recombination
frequency
TABLE II ______________________________________ Frequency of
rearrangements in three cell lines Amp.sup.R colonies R = 3 .times.
Blue .times. 10.sup.-2 Cell line DNA Total Blue Total
______________________________________ A9 pBlueRec, pRag1, 16,800 0
0 pRag2 pBlueRec, pRag1, 5,620 71 3.8 pRag2 BW1J pBlueRec 12,000 0
0 pBlueRec, pRag1, 5,600 83 4.4 pRag2 pBlueRec, pRag1, 1,800 8 1.3
pRag2 pBlueRec 16,000 1050 19.6 CHO-K1 pBlueRec 3,434 0 0 pBlueRec,
pRag1, 2,300 38 4.9 pRag2 pBlueRec, pRag1, 1,996 149 22.3 pRag2
______________________________________ R = recombination
frequency
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 62 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CCGCTCTAGAACTAGTGGATCCCACAGTGCACTGTGGTCGACCTCGAGGGG51 (2)
INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:2:
CCGCTCTAGAACTAGTGGATACCTCGAGGGG31 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:3: CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2) INFORMATION FOR
SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:4: CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2)
INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:5:
CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2) INFORMATION FOR SEQ ID
NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:6: CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2) INFORMATION FOR
SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:7: CCGCTCTAGAACTAGTGGATCCGACCTCGAGGGG34 (2) INFORMATION
FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:8: CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2)
INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:9:
CCGCTCTAGAACTAGTGGATCCGACCTCGAGGGG34 (2) INFORMATION FOR SEQ ID
NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:10: CCGCTCTAGAACTAGTGGCGACCTCGAGGGG31 (2) INFORMATION FOR SEQ ID
NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:11: CCGCTCTAGAACTAGTGGATCCGACCTCGAGGGG34 (2) INFORMATION FOR SEQ
ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:12: CCGCTCTAGAACTAGTGTCGAGGGG25 (2) INFORMATION FOR SEQ
ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:13: CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2)
INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:14:
CCGCTCTAGAACTAGTGGATCCGACCTCGAGGGG34 (2) INFORMATION FOR SEQ ID
NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:15: CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2) INFORMATION FOR
SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:16: CCGCTCTAGAACTAGTGGATCCGGCGACCTCGAGGGG37 (2)
INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:17:
CCGCTCTAGAACTAGTGGCGACCTCGAGGGG31 (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:18: CCGCTCTAGAACTAGTGGATCCCTCGACCTCGAGGGG37 (2) INFORMATION FOR
SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:19: CCGCTCTAGAACTAGTGGGTTCGTCGACCTCGAGGGG37 (2)
INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:20:
CCGCTCTAGAACTAGTGGATCCGGCCTCGAGGGG34 (2) INFORMATION FOR SEQ ID
NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:21: CCGCTCTAGAACTAGTGGGGCCGTCGACCTCGAGGGG37 (2) INFORMATION FOR
SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:22: CCGCTCTTTCGTCGACCTCGAGGGG25 (2) INFORMATION FOR SEQ
ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:23: CCGCTCTAGAACTAGTTTCCTCGAGGGG28 (2) INFORMATION FOR
SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:24: CCGCTCTAGAACTAGTGGGGTCGACCTCGAGGGG34 (2) INFORMATION
FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:25: CCGCTCTAGAACTAGTGGATCCCCAGACCTCGAGGGG37 (2)
INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:26: CCGCTCTAGAACTTTCGACCTCGAGGGG28
(2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:27:
CCGCTCTAGAACTAGTGGATCCGACGTCGACCTCGAGGGG40 (2) INFORMATION FOR SEQ
ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
CCGCTCTAGAACTAGTGGATCCATCCGACCTCAGGGG37 (2) INFORMATION FOR SEQ ID
NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:29: CCGCTCTAGAACTAGTGGATCCAACCTCGAGGGG34 (2) INFORMATION FOR SEQ
ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:30: CCGCTCTAGAACTAGTGACCTCGAGGGG28 (2) INFORMATION FOR
SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:31: CCGCTCTAGAACTAGTGGATCCTCCGTCGACCTCGAGGGG40 (2)
INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:32:
CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2) INFORMATION FOR SEQ ID
NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:33: CCGCTCTAGAACTAGTGGATCCCTCGTCGACCTCGAGGGG40 (2) INFORMATION
FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:34: CCGCTCTAGAACTAGTGGGTCCGTCGACCTCGAGGGG37 (2)
INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:35:
CCGCTCTAGAACTAGTGGATCGGGCCTCGAGGGG34 (2) INFORMATION FOR SEQ ID
NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:36: CCGCTCTAGAACTAGTGGATCCGAGGTCGACCTCGAGGGG40 (2) INFORMATION
FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:37: CCGCTCTAGAACTAGTGGATCGGACCTCGAGGGG34 (2) INFORMATION
FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:38: CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2)
INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 52 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:39:
CCGCTCTAGAACTAGTGGATACCATACCCCTTTACCAATCGACCTCGAGGGG52 (2)
INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:40:
CCGCTCGAGAACTAGTGGATCCCCCCCCGCCGTCGACCTCGAGGGG46 (2) INFORMATION
FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:41: CCGCTCTAGAACTAGTGGTCCTCCTCGAGGGG32 (2) INFORMATION
FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:42: CCGCTCTAGAACTAGTGGATCACACCTCGAGGGG34 (2) INFORMATION
FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:43: CCGCTCTAGAACTAGTGGCCGACCTCGAGGGG32 (2) INFORMATION
FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:44: CCGCTCTAGAACTAGCCCTACCGACCTCGAGGGG34 (2) INFORMATION
FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:45: CCGCTCTAGAACTAGTGGCCCCGACCTCGAGGGG34 (2) INFORMATION
FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:46: CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2)
INFORMATION FOR SEQ ID NO:47: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:47:
CCGCTCTAGAACTAGTGGTCCCGACCTCGAGGGG34 (2) INFORMATION FOR SEQ ID
NO:48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:48: CCGCTCTAGAACTAGTGGATTCGTCGACCTCGAGGGG37 (2) INFORMATION FOR
SEQ ID NO:49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:49: CCGCTCTAGAACTAGTGGTCTCGACCTCGAGGGG34 (2) INFORMATION
FOR SEQ ID NO:50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:50: CCGCTCTAGAACTAGTGGATCCAACCTCGAGGGG34 (2) INFORMATION
FOR SEQ ID NO:51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:51: CCGCTCTAGAACTAGTGGATCCCCCGTCGACCTCGAGGGG40 (2)
INFORMATION FOR SEQ ID NO:52: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:52:
CCGCTCTAGAACTAGTGGGACCTCGAGGGG30 (2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:53: CCGCTCTAGAACTAGTGGACCTCGAGGGG29 (2) INFORMATION FOR SEQ ID
NO:54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:54: CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2) INFORMATION FOR
SEQ ID NO:55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:55: CCGCTCTAGAACTAGTGGATCCGACCTCGAGGGG34 (2) INFORMATION
FOR SEQ ID NO:56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base
pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:56: CCGCTCTAGAACTAGTGGATCCGACCTCGAGGGG34 (2) INFORMATION
FOR SEQ ID NO:57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:57: CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2)
INFORMATION FOR SEQ ID NO:58: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:58:
CCGCTCTAGAACTAGTGGATCCGGCCTCGAGGGG34 (2) INFORMATION FOR SEQ ID
NO:59: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:59: CCGCTCTAGAACTAGTGGATCCGTCGACCTCGAGGGG37 (2) INFORMATION FOR
SEQ ID NO:60: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:60: CCGCGACCTCGAGGGG16 (2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:61: CCGCGTCGACCTCGAGGGG19 (2) INFORMATION FOR SEQ ID NO:62: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:62: CCGCTCTAGAACTAGTGGATCGACCTCGAGGGG33
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